Alteration of chromatin by covalent modification of histone proteins is central to regulation of gene expression. Acetylation, the best-characterized covalent modification of histones, has wide-ranging effects on gene expression. Histone phosphorylation is important for transcriptional activation, condensation of chromosomes during mitosis and meiosis, and regulation of cell division. Roles for histone methylation and monoubiquitination in the regulation of gene expression have recently been revealed. For several years we have been studying the role of the MLL protein in the development of human leukemia. Setl protein of S. cerevisiae is closely related to human MLL. We and others have purified a Setl containing multiprotein complex that we call COMPASS. We have demonstrated that COMPASS is required for methylation of lysine 4 in the amino-terminal tail of histone H3, and that this histone modification is required for silencing of expression of genes located near chromosome telomeres. We found that recruitment of COMPASS occurs preferentially at the promoter and with the early elongating polymerase. In a survey of the yeast genome for gene products necessary for histone H3 methylation, we discovered that the ubiquitin-conjugating enzyme Rad6, together with its likely E3 ligase, Bre1, and the Paf1 complex are required for methylation of lysine 4 and lysine 79 of histone H3. Since Rad6 catalyzes monoubiquitination of lysine 123 of histone H2B, this modification of histone H2B may be a signal that directs COMPASS to catalyze methylation of K4 of histone H3. We wish to define the mechanism of histone ubiquitination by Rad6, Brel, and the Pafl complex and their roles in regulation of gene expression via histone modifications by monoubiqutination. Our specific aims are: Aim 1: To understand how monoubiquitination of lysine 123 of histone H2B by Rad6/Bre1 is limited to the promoter regions of genes, and learn about the consequences of this event for gene regulation. Aim 2: To learn if monoubiquitination of histone H2B serves as a signal for the recruitment and/or activation of the histone methyltransferase COMPASS. Aim 3: To define the molecular mechanism ofdeubiquitination of historic H2B. These studies promise to: i) improve our understanding of the role of histone monoubiquitination in regulation of transcription, ii) shed light on how and why ubiquitination occurs preferentially at promoters, iii) reveal if, why, and how ubiquitination of histones at promoters signals histone methylation, and iv) shed light on the role of the conserved Set domain in yeast Setl and human MLL in development and pathogenesis of hematological malignancies.